One of the principal mechanisms by which cellular regulation is mediated is through the transformation of extracellular signals into intracellular signals that in turn modulate biochemical pathways. Cell surface receptors are one of the principal means by which extracellular regulatory information is transmitted to the cell. Cellular phenotypes are therefore largely influenced by the normal functioning of extracellular ligand/cell surface receptor interactions. Consequently, it is currently believed that a number of disease states and/or disorders are a result of aberrant expression or mutations in cell surface molecules that mediate communication between cells and their surrounding environment. In recent years, a number of these cell surface receptors have been cloned and their functions characterized. Among these is a ubiquitously expressed, multifunctional cell surface adhesion receptor known as CD44.
A number of extracellular matrix (ECM) and non-ECM molecules have been identified as ligands for CD44, including hyaluronic acid, fibronectin, laminin, collagen, and chondroitin sulfate. Non-ECM ligands for CD44 include mucosal adressin, serglycin, and osteopontin. The ability of CD44 to interact with multiple ligands has been explained in part by the identification thus far of over twenty distinct CD44 isoforms (Naor et al., Adv. Cancer Res., 1997, 71, 241-319). Ranging from 85-230 kDa, CD44 isoforms have been found to differ in both expression patterns and functional properties. The generation of CD44 isoforms is due to variability in mRNA splicing and posttranslational processing. The CD44 gene is composed of twenty exons. The first five and last five exons are constant and as such are present in all CD44 isoforms, whereas the middle 10 exons comprise the variable region that is subject to alternative splicing. The smallest CD44 isoform, known as CD44 standard (CD44s), lacks the entire variable region and is expressed mainly on hematopoietic cells. The longest CD44 isoform identified, expressed by keratinocytes, contains exons 3 through 10 of the variable region (CD44V3-10).
CD44 participates in a relatively diverse set of functions including cell-cell and cell-matrix interactions, cell trafficking, lymph node homing, presentation of chemokines and growth factors to cells, and transmission of signals involved in mitogenesis, hematopoiesis, and apoptosis (Naor et al., Adv. Cancer Res., 1997, 71, 241-319). A number of diseases have been linked to aberrant CD44 expression and/or splicing as well, including rheumatoid arthritis and cancers of the breast, gastrointestinal tract, uterus, bladder, lung, brain, and hematopoietic tissue (Cooper, J. Pathol., 1995, 177, 1-3; Naor et al., Adv. Cancer Res., 1997, 71, 241-319; Tarin et al., J. Neurooncol., 1995, 26, 209-219).
Initial evidence linking CD44 with rheumatoid arthritis was provided by several reports documenting elevated CD44 expression on rheumatoid synovial cells. Subsequently, a number of studies have demonstrated reductions in joint swelling, edema, and leukocyte infiltration by monoclonal anti-CD44 antibodies in murine models of proteoglycan and collagen induced arthritis (Naor et al., Adv. Cancer Res., 1997, 71, 241-319). Gunthert et al. demonstrated that transfection of rat carcinoma cells with a CD44 splice variant conferred metastatic potential, suggesting a role for CD44 in tumor progression (Gunthert et al., Cell, 1991, 65, 13-24). In support of this finding, a number of malignancies and their metastatic lesions have since been shown to express highly elevated levels of CD44 and/or novel splice variants, which has led to the notion that CD44 may serve as a useful prognostic marker (Cooper, J. Pathol., 1995, 177, 1-3). Finally, recent studies in animal models have demonstrated inhibition of tumor growth and metastatic spread following injection of compounds that interfere with CD44-ligand interactions, suggesting that expression of CD44 provides a growth advantage to certain neoplastic cell types (Naor et al., Adv. Cancer Res., 1997, 71, 241-319; Tarin et al., J. Neurooncol., 1995, 26, 209-219).
Antisense inhibition of CD44 has been used to elucidate the role of CD44. Kaya et al. describe the development of transgenic mice that express CD44 in antisense orientation and report that inhibition of CD44 in keratinocytes is accompanied by alterations in skin elasticity, local inflammatory response, tissue repair, hair growth, and hyperplasia in response to carcinogen treatment (Kaya et al., Genes Dev, 1997, 11, 996-1007). Lamb et al. disclose the use of antisense oligodeoxynucleotides in transformed rat fibroblasts and find that CD44 antisense oligonucleotide treatment is accompanied by inhibition of invasion through a collagen matrix gel (Lamb et al., Mol Cell Biol, 1997, 17, 963-976). WO97/06827 discloses antisense oligonucleotide sequences directed against the human CD44 gene capable of inhibiting in vitro growth of a lung carcinoma cell line (Pietrzkow et al., 1997). Merzak et al. disclose a CD44 antisense oligonucleotide targeted to a sequence starting at position 1725 of the human CD44 sequence published by Stamenkovic et al. (Merzak et al., Cancer Res, 1994, 54, 3988-3992; Stamenkovic et al., Embo J, 1991, 10, 343-348). The oligonucleotide was able to inhibit invasion of human glioma cells through a collagen matrix gel (Merzak et al., Cancer Res, 1994, 54, 3988-3992). Zhu et al. disclose liposome mediated delivery of the same oligonucleotide to inhibit CD44 expression and to interfere with the invasive behavior of glioma cells (Zhu et al., Targeting of Drugs 5: Strategies for oligonucleotide and gene delivery in therapy., 1996, 169-178). Chow et al. disclose antisense oligonucleotides directed against bovine CD44 mRNA and report that treatment of bovine cartilage tissue slices with these compounds disrupt proteoglycan homeostasis (Chow et al., Arthritis Rheum, 1998, 41, 1411-1419). Finally, Reeder et al. describe the generation of an HT-29 colorectal cell line stably expressing exon 10 of the human CD44 gene in antisense orientation. Following injection into nude mice, antisense expressing HT-29 cells, compared to untransfected or vector-only transfected cells, were found to have a lower incidence of liver metastasis formation and tumor formation at sites where incisional wounds were introduced (Reeder et al., Cancer Res, 1998, 58, 3719-3726).
There remains a long felt need for improved compositions and methods for inhibiting CD44 gene expression.